Biomedical subjects

Christopher M Counter

Publications and source records attributed to Christopher M Counter.

At least 19 recordsLinked to original sources

PubMed2026

FANCM is required for the PAX3::FOXO1-driven oncogenic program in rhabdomyosarcoma.

Many cancers are driven by mutationally altered transcription factors (TFs) that rewire cells to an oncogenic state. Cells must activate specific mechanisms to tolerate the burden of oncogenic TF activity. To define such mechanisms, we focused on a canonical oncogenic fusion protein-driven cancer, alveolar rhabdomyosarcoma (ARMS), where the PAX3::FOXO1 fusion protein hyperactivates and mislocalizes PAX3 and FOXO1 TF functions. Employing sequential functional genomic CRISPR-Cas9 screens, we identified FANCM, a DNA translocase in the Fanconi anemia pathway, as a selective dependency in PAX3::FOXO1+ ARMS. FANCM loss reduces fusion protein levels, induces myogenic differentiation, and disrupts the PAX3::FOXO1 transcriptional program, thereby halting oncogenic proliferation. Mechanistically, FANCM depletion exacerbates replication stress (RS) and DNA damage signaling, with chromatin-associated RS enriched at PAX3::FOXO1 target gene loci, resulting in selective downregulation of the oncogenic program. CRISPR exon-tiling screens prioritized FANCM's helicase and DNA-binding domains as essential for this dependency, linking FANCM-mediated replication fork binding to sustained oncogenesis.

ARMS↗

PubMed2006

Divergent roles for RalA and RalB in malignant growth of human pancreatic carcinoma cells.

BACKGROUND: The Ral guanine nucleotide-exchange factors (RalGEFs) serve as key effectors for Ras oncogene transformation of immortalized human cells. RalGEFs are activators of the highly related RalA and RalB small GTPases, although only the former has been found to promote Ras-mediated growth transformation of human cells. In the present study, we determined whether RalA and RalB also had divergent roles in promoting the aberrant growth of pancreatic cancers, which are characterized by the highest occurrence of Ras mutations. RESULTS: We now show that inhibition of RalA but not RalB expression universally reduced the transformed and tumorigenic growth in a panel of ten genetically diverse human pancreatic cancer cell lines. Despite the apparent unimportant role of RalB in tumorigenic growth, it was nevertheless critical for invasion in seven of nine pancreatic cancer cell lines and for metastasis as assessed by tail-vein injection of three different tumorigenic cell lines tested. Moreover, both RalA and RalB were more commonly activated in pancreatic tumor tissue than other Ras effector pathways. CONCLUSIONS: RalA function is critical to tumor initiation, whereas RalB function is more important for tumor metastasis in the tested cell lines and thus argues for critical, but distinct, roles of Ral proteins during the dynamic progression of Ras-driven pancreatic cancers.

Adenocarcinoma↗

PubMed2006

Genetically engineered human cancer models utilizing mammalian transgene expression.

Cancer models are vital to cancer biology research, and multiple cancer models are currently available that utilize either murine or human cells, each with particular strengths and weaknesses. The ability to transform primary human cells into tumors through the expression of specific transgenes offers many advantages as a cancer model, including genetic malleability and the ability to transform specific cell types. Until recently, the conversion of primary human cells into tumors through transgene expression required the use of viral genetic elements, which unfortunately adds uncertainty regarding which cancer pathways are affected and how they are affected. In recent years multiple reports have described the transformation of primary human cells into tumors using only mammalian transgenes. This review focuses on these five cancer models, comparing the different cell types which were transformed into tumors and which transgenes were expressed, as well as the cancer pathways affected in the disparate models. These genetically-engineered human cancer models offer a valuable tool to complement existing cancer models and further cancer research.

Animals↗

PubMed2006

hSnm1B is a novel telomere-associated protein.

Artemis, a member of the beta-CASP family, has been implicated in the regulation of both telomere stability and length. Prompted by this, we examined whether the other two putative DNA-binding members of this family, hSnm1A and hSnm1B, may associate with telomeres. hSnm1A was found to not interact with the telomere. Conversely, hSnm1B was found to associate with telomeres in vivo by both immunofluorescence and chromatin immunoprecipitation. Furthermore, the C terminus of hSnm1B was shown to interact with the TRF homology domain of TRF2 indicating that hSnm1B is likely recruited to the telomere via interaction with the double-stranded telomere-binding protein TRF2.

Binding Sites↗

PubMed2006

Use of retrovirus expression of interfering RNA to determine the contribution of activated K-Ras and ras effector expression to human tumor cell growth.

Cancer is a multistep genetic process that includes mutational activation of oncogenes and inactivation of tumor suppressor genes. The Ras oncogenes are the most frequently mutated oncogenes in human cancers (30%), with a high frequency associated with cancers of the lung, colon, and pancreas. Mutational activation of Ras is commonly an early event in the development of these cancers. Thus, whether mutated Ras is required for tumor maintenance and what aspects of the complex malignant phenotype might be promoted by mutated Ras are issues that remain unresolved for these and other human cancers. The recent development of interfering RNA to selectively impair expression of mutated Ras provides a powerful approach to begin to resolve these issues. In this chapter, we describe the use of retrovirus-based RNA interference approaches to study the functions of Ras and Ras effectors (Raf, RalA, RalB, and Tiam1) in the growth of pancreatic carcinoma and other human tumor cell lines. Finally, we also compare the use of constitutive and inducible shRNA expression vectors for analyses of mutant Ras function.

Base Sequence↗

PubMed2006

A genetically defined normal human somatic cell system to study ras oncogenesis in vivo and in vitro.

Transgenic mice, cultured murine cells, and human cancer cell lines have widely been used to study Ras oncogenesis. Although extremely valuable systems, they could not be used to study Ras function in genetically defined human cells. In this regard, Ras is required for tumor formation in normal human somatic cells expressing SV-40 T/t antigens, which inactivate the tumor suppressors p53 and Rb and activate the oncogene c-Myc, and hTERT, the catalytic subunit of telomerase. Such a system allows not only the general requirements of Ras to be dissected in matched cells from different organisms or tissues but also the individual pathways required for tumor growth to be defined in human cells. This review will detail the methods of creating stable T/t Ag, TERT, Ras-expressing cell lines, as well as commonly used techniques of soft agar and xenograft tumor formation.

Animals↗

PubMed2005

A network of genetic events sufficient to convert normal human cells to a tumorigenic state.

Although great progress has been made at identifying and characterizing individual genes involved in cancer, less is known about how the combination of such genes collaborate to form tumors in humans. To this end, we sought to genetically recreate tumorigenesis in normal human cells using genes altered in human cancer. We now show that expression of mammalian proteins that inactivate the tumor suppressors Rb and p53 in conjunction with the oncoproteins Ras and Myc and the telomerase subunit hTERT is sufficient to drive a number of normal human somatic cells to a tumorigenic fate. This provides a blueprint of the events that lead to human cancer, allowing different cancers to be genetically modeled from normal human cells.

Animals↗

PubMed2005

Genetic modeling of human rhabdomyosarcoma.

Rhabdomyosarcoma, a malignancy showing features of skeletal muscle differentiation, is the most common soft tissue sarcoma of childhood. The identification of distinct clinical presentation patterns, histologic tumor types, and risk groups suggests that rhabdomyosarcoma is a collection of highly related sarcomas rather than a single entity. In an effort to understand this seemingly heterogeneous malignancy, we constructed a genetically defined but malleable model of rhabdomyosarcoma by converting less differentiated human skeletal muscle cell precursors (SkMC) and committed human skeletal muscle myoblasts (HSMM) into their malignant counterparts by targeting pathways altered in rhabdomyosarcoma. Whereas the two cell types were both tumorigenic, SkMCs gave rise to highly heterogeneous tumors occasionally displaying features of rhabdomyosarcoma, whereas HSMMs formed rhabdomyosarcoma-like tumors with an embryonal morphology, capable of invasion and metastasis. Thus, despite introducing the same panel of genetic changes, altering the skeletal muscle cell of origin led to different tumor morphologies, suggesting that cell of origin may dictate rhabdomyosarcoma tumor histology. The ability to now genetically induce human rhabdomyosarcoma-like tumors provides a representative model to dissect the molecular mechanisms underlying this cancer.

Adolescent↗

PubMed2005

Activation of RalA is critical for Ras-induced tumorigenesis of human cells.

RalGEFs were recently shown to be critical for Ras-mediated transformed and tumorigenic growth of human cells. We now show that the oncogenic activity of these proteins is propagated by activation of one RalGEF substrate, RalA, but blunted by another closely related substrate, RalB, and that the oncogenic signaling requires binding of the RalBP1 and exocyst subunit effector proteins. Knockdown of RalA expression impeded, if not abolished, the ability of human cancer cells to form tumors. RalA was also commonly activated in a panel of cell lines from pancreatic cancers, a disease characterized by activation of Ras. Activation of RalA signaling thus appears to be a critical step in Ras-induced transformation and tumorigenesis of human cells.

ATP-Binding Cassette Transporters↗

PubMed2005

Reduction in the requirement of oncogenic Ras signaling to activation of PI3K/AKT pathway during tumor maintenance.

While tumors become addicted to oncogenes like Ras, the microenvironment in which tumor cells reside changes during tumorigenesis; the cells are surrounded initially by normal tissue and later by tumor tissue. Hence, we asked if Ras exerts its oncogenic effects through the same set of effectors during different stages of tumorigenesis. We now show in human cells that the Ras effector pathways MAPK, RalGEF, and PI3K are required to initiate tumor growth. Conversely, activation of the PI3K/AKT pathway replaced Ras once tumors formed, although other effectors were still activated independently of Ras, presumably by factors provided upon the establishment of a tumor microenvironment. Thus, as tumorigenesis progresses the addiction of cancers to their initiating oncogene is reduced to, at least in the case of Ras, the PI3K/AKT pathway.

Animals↗

PubMed2004

Loss of hPot1 function leads to telomere instability and a cut-like phenotype.

The human telomere binding protein hPot1 binds to the most distal single-stranded extension of telomeric DNA in vitro, and probably in vivo, as well as associating with the double-stranded telomeric DNA binding proteins TRF1 and TRF2 through the bridging proteins PTOP (also known as PIP1 or TINT1) and TIN2. Disrupting either the DNA binding activity of hPot1 or its association with PTOP results in elongated telomeres, suggesting a role for hPot1 in telomere length regulation. However, mutations to POT1 and Cdc13p, the fission and budding yeast genes encoding the structural orthologs of this protein, leads to telomere instability and cell death. Thus, it is possible that the hPot1 protein may also serve to cap and protect telomeres in humans. Indeed, we now find that knocking down the expression of hPot1 in human cells causes apoptosis or senescence, as well as an increase in telomere associations and anaphase bridges, telltale signs of telomere instability. In addition, knockdown cells also displayed chromatin bridges between interphase cells, reminiscent of the cut phenotype that was first described in fission yeast and in which cytokinesis progresses despite a failure of chromatid separation. However, unlike the yeast cut phenotypes, we suggest that the cut-like phenotype observed in hPot1 knockdown cells is a consequence of the fusion of chromosome ends and that this fusion impedes proper chromosomal segregation. We conclude that hPot1 protects chromosome ends from illegitimate recombination, catastrophic chromosome instability, and abnormal chromosome segregation.

Apoptosis↗

PubMed2004

Characterization of interactions between PinX1 and human telomerase subunits hTERT and hTR.

The addition of telomeric repeats to chromosome ends by the enzyme telomerase is a highly orchestrated process. Although much is known regarding telomerase catalytic activity in vitro, less is known about how this activity is regulated in vivo to ensure proper telomere elongation. One protein that appears to be involved in negatively regulating telomerase function in vivo is PinX1 because overexpression of PinX1 inhibits telomerase activity and causes telomere shortening. To understand the nature of this repression, we characterized the interactions among PinX1 and the core components of telomerase, the human telomerase reverse transcriptase (hTERT) and associated human telomerase RNA (hTR). We now show that in vitro PinX1 binds directly to the hTERT protein subunit, primarily to the hTR-binding domain, as well as to the hTR subunit. However, in a cellular context, the association of PinX1 with hTR is dependent on the presence of hTERT. Taken together, we suggest that PinX1 represses telomerase activity in vivo by binding to the assembled hTERT.hTR complex.

Cell Cycle Proteins↗

PubMed2004

Leveling the playing field.

Using a genetically malleable model system that allows direct comparisons between human and mouse cells, Weinberg and colleagues lay to rest any doubt that murine cells are more easily transformed than human cells, and additionally, find that there may be cell type constraints on transformation of human cells.

Animals↗

PubMed2004

A signalling pathway controlling c-Myc degradation that impacts oncogenic transformation of human cells.

The stability of c-Myc is regulated by multiple Ras effector pathways. Phosphorylation at Ser 62 stabilizes c-Myc, whereas subsequent phosphorylation at Thr 58 is required for its degradation. Here we show that Ser 62 is dephosphorylated by protein phosphatase 2A (PP2A) before ubiquitination of c-Myc, and that PP2A activity is regulated by the Pin1 prolyl isomerase. Furthermore, the absence of Pin1 or inhibition of PP2A stabilizes c-Myc. A stable c-Myc(T58A) mutant that cannot bind Pin1 or be dephosphorylated by PP2A replaces SV40 small T antigen in human cell transformation and tumorigenesis assays. Therefore, small T antigen, which inactivates PP2A, exerts its oncogenic potential by preventing dephosphorylation of c-Myc, resulting in c-Myc stabilization. Thus, Ras-dependent signalling cascades ensure transient and self-limiting accumulation of c-Myc, disruption of which contributes to human cell oncogenesis.

Amino Acid Sequence↗

PubMed2004

Rescue of an hTERT mutant defective in telomere elongation by fusion with hPot1.

The protein hPot1 shares homology with telomere-binding proteins in lower eukaryotes and associates with single-stranded telomeric DNA in vitro as well as colocalizing with telomere-binding proteins in vivo. We now show that hPot1 is coimmunoprecipitated with telomeric DNA and that stable expression of this protein in telomerase-positive cells results in telomere elongation, supporting the idea that hPot1 is a bona fide mammalian telomere-binding protein. We previously found that mutations in the N-terminal DAT domain of the hTERT catalytic subunit of telomerase rendered the enzyme catalytically active but unable to elongate telomeres in vivo. This phenotype could be partially rescued by fusion with the double-stranded telomeric protein hTRF2. Given that hPot1 binds to single-stranded DNA in vitro (at the same site that hTERT binds to in vivo), we addressed whether fusion of hPot1 can rescue the DAT mutations more efficiently than that of hTRF2. We now report that a DAT mutant of hTERT is indeed efficiently rescued upon fusion to hPot1. However, this rescue depended on the ability of hPot1 to localize to telomeres rather than binding to DNA per se. These data support a model whereby the DAT domain of hTERT is implicated in telomere-telomerase associations.

Amino Acid Sequence↗

PubMed2004

Genomics and clinical medicine: rationale for creating and effectively evaluating animal models.

Because resolving human complex diseases is difficult, appropriate biomedical models must be developed and validated. In the past, researchers have studied diseases either by characterizing a human clinical disease and choosing the most appropriate animal model, or by characterizing a naturally occurring or induced mutant animal and identifying which human disease it best resembled. Although there has been a great deal of progress through the use of these methods, such models have intrinsic faults that limit their relevance to clinical medicine. The recent advent of techniques in molecular biology, genomics, transgenesis, and cloning furnishes investigators with the ability to study vertebrates (e.g., pigs, cows, chickens, dogs) with greater precision and utilize them as model organisms. Comparative and functional genomics and proteomics provide effective approaches for identifying the genetic and environmental factors responsible for complex diseases and in the development of prevention and treatment strategies and therapeutics. By identifying and studying homologous genes across species, researchers are able to accurately translate and apply experimental data from animal experiments to humans. This review supports the hypothesis that associated enabling technologies can be used to create, de novo, appropriate animal models that recapitulate the human clinical manifestation. Comparative and functional genomic and proteomic techniques can then be used to identify gene and protein functions and the interactions responsible for disease phenotypes, which aids in the development of prevention and treatment strategies.

Animals↗

PubMed2003

Telomere shortening in cultured autografts of patients with burns.

In extensive third-degree burns, donor sites for conventional split thickness skin grafts are limited. In such cases, cultured epithelial (keratinocyte) grafts are prepared from small samples of the patient's own skin and expanded in tissue culture, a process that may incur very many cell divisions. Telomeres shorten with each cell division, and are markers of cellular proliferative history. We therefore measured telomere length in healed cultured epithelial autografts from four patients with burns, and noted that their telomeres were shorter than those in non-cultured skin from the same individuals, and than those in skin of healthy donors older than 80 years. Such great loss of telomeric DNA suggests that engrafted cells might have a shortened lifespan, which could have negative repercussions on the long-term viability of these grafts.

Aged↗

PubMed2003

Bone-related genes expressed in advanced malignancies induce invasion and metastasis in a genetically defined human cancer model.

We employed a genetically defined human cancer model to investigate the contributions of two genes up-regulated in several cancers to phenotypic changes associated with late stages of tumorigenesis. Specifically, tumor cells expressing two structurally unrelated bone-related genes, osteonectin and osteoactivin, acquired a highly invasive phenotype when implanted intracranially in immunocompromised mice. Mimicking a subset of gliomas, tumor cells invaded brain along blood vessels and developed altered vasculature at the brain-tumor interface, suggesting that production of those two proteins by tumor cells may create a complex relationship between invading tumor and vasculature co-opted during tumor invasion. Interestingly, the same tumor cells formed massive spontaneous metastases when implanted subcutaneously. This dramatic alteration in tumor phenotype indicates that cellular microenvironment plays an important role in defining the specific effects of those gene products in tumor behavior. In vitro examination of tumor cells expressing either osteonectin or osteoactivin revealed that there was no impact on cellular growth or death but increased invasiveness and expression of MMP-9 and MMP-3. Specific pharmacologic inhibitors of MMP-2/9 and MMP-3 blocked the increased in vitro invasion associated with osteoactivin expression, but only MMP-3 inhibition altered the invasive in vitro phenotype mediated by osteonectin. Results from this genetically defined model system are supported by similar findings obtained from several established tumor cell lines derived originally from human patients. In sum, these results reveal that the expression of a single bone-related gene can dramatically alter or modify tumor cell behavior and may confer differential growth characteristics in different microenvironments. Genetically defined human cancer models offer useful tools in functional genomics to define the roles of specific genes in late stages of carcinogenesis.

Animals↗